The objective of this
program is the continued exploration of gene therapy procedures to introduce
functional portions of the insulin-like growth factor-I (IGF-I) gene
into joints damaged by OCD, acute injury, or those in the early stages
of arthritis. Growth factors, particularly IGF-I, are predominantly
involved in the maintenance of healthy cartilage by stimulating cartilage
cell metabolism. After injury, this cartilage homeostatic balance is
perturbed by a proliferation of degradatory enzymes and other bioactive
peptides which insidiously damage the cartilage structure. The restoration
of this balance normally depends on reduced exercise, surgical intervention,
oral anti-inflammatory and pain relieving agents, and extended periods
of rest. Untreated or severely damaged joints frequently develop osteoarthritis
which remains a leading cause of retirement of horses from active racing
and often precludes even modest exercise programs. Enhanced levels of
stimulatory growth factors such as IGF-I can be experimentally provided
by articular injection or the use of slow-release polymers. However,
both result only in short periods of growth factor exposure, with little
possibility of long-term impact on the joint. Methods to permanently
enhance growth factor articular concentrations are being explored in
this grant and utilize previous work on genetically engineered equine
IGF-I constructs which will be introduced to joints by viral vectors,
resulting in incorporation of the IGF-I gene into the cell nuclei of
joint lining and cartilage cells. Despite recent untoward experiences
in several human systemic gene therapy trials, the use of local gene
therapy protocols, such as local joint delivery, remains a safe and
effective mechanism to improve joint health in the long-term.

Our previous Zweig funded studies have
cloned and sequenced both equine IGF-I and transforming growth
factor-beta (TGF-beta). These gene products produce IGF-I and
TGF-beta proteins that have been extensively evaluated in equine
tissue culture systems. Further, our evaluation of the expression
of these growth factors after cartilage injury shows that an
early deficiency is followed by a transitory peak at 8 weeks,
only to decline again at 16 weeks and beyond. This information
indicates an early and a late window of opportunity when supplemental
endogenous IGF-I or TGF-beta may be particularly useful in improving
cartilage repair. Our studies suggest TGF-beta enhances cellular
division among chondrocytes and stem cells, but has a limited
potential to drive up cartilage matrix synthesis. Fortunately,
IGF-I has largely complementary activity, with minimal effects
on cell division, but a significant impact on matrix proliferation.
As a result, selection of IGF-I may be useful when chondrocytes
are already present in adequate numbers, while TGF-beta may have
an earlier application in deep cartilage injuries when numbers
of differentiated cartilage cells are inadequate. Application
of composites of IGF-I and chondrocytes to cartilage repair has
resulted in significant joint regeneration, and now represents
common clinical practice in the equine hospital of the Cornell
University Hospital for Animals. However, longevity of the IGF-I
impact on transplant chondrocyte activity is limited by protein
residence. This grant seeks to examine the effect of inserting
the gene for IGF-I into chondrocytes at the time of implantation,
which is proposed to extend the impact of IGF-I on the anabolic
activity of the new chondrocytes beyond the 2 weeks previously
reported in trials of IGF-I protein laden chondrocyte transfers.

This experiment continues a series of
trials evaluating biologic delivery mechanisms to transport the
active portion of equine growth factor genes to joint structures.
Studies in 1999 showed that the IGF-I gene can be inserted into
chondrocytes and synovial cells forming the lining of joints,
and that once inside cells the gene produces active IGF-I protein
for as long as 30 days. In vivo studies performed in 2000 confirmed
that the equine IGF-I gene could be directly injected to the
fetlock and take up residence by seeding the joint synovial lining.
Expression of IGF-I was found in joint fluid over 90 days, which
would provide significant prolonged effects on healing cartilage.
These gene transfer experiments use a viral piggyback system,
where the gene coding IGF-I "infects" cells forming
the interior lining of the knee joint. The combination of the
equine IGF-I gene and a modified adenovirus used simple gene
splicing techniques to yield a virion particle capable of penetrating
living cells and delivering IGF-I DNA to the host cell genome.

These trials suggested that the adenovirus
achieved high incorporation rates, and provided a solid foundation
for additional in vivo testing of adeno-IGF-I in equine joints.
Clinically, the adenoviral construct does have a major practical
advantage in that it can be administered to a joint by injection,
thereby providing a relatively non-invasive method for growth
factor gene delivery. Gene enhanced chondrocytes for use in
transplant systems is the logical next step, since this would
allow local autostimulation of cartilage matrix synthesis in
newly grafted cartilage defects. Subsequent followup joint injections
of further vector would then bolster chondrocyte function via
synovial seeding and IGF-I production which would enter the joint
fluid and influence cartilage healing by diffusion. Our year
2000 in vivo studies show the adenoviral-IGF-I vector provides
a quick "hit" to joints that is simple to administer
by injection. The proposed study for 2001 tests the hypothesis
that adeno-IGF-I transfer to chondrocytes at the time of implantation
will enhance chondrocyte anabolic functions sufficiently to drive
significant new cartilage repair, Such a system is a natural
progression for the current chondrocyte transplant program, and
actually simplifies that protocol by eliminating the need for
IGF-I addition to the milieu at implant. Rather, the cells will
make their own IGF-I; a simple, safe and perhaps more efficacious
means to bolster cartilage healing.

To test this hypothesis, the present
proposal plans a study in the horse stifle, where cartilage defects
are either filled with IGF-I gene enhanced chondrocytes or chondrocytes
exposed to a null gene in the same vector. The healing response
will be evaluated by arthroscopic examination and biopsy at 1
and 2 months after implant, and final tissue analysis 8 months
after repair. Persistence of the IGF-I gene at each time point
will be determined by sensitive PCR techniques, which will determine
whether additional direct injection of IGF-I gene vectors to
the joint fluid will be required in clinical cases. Such a dual
approach combines previous year 2000 study results with current
proposals in a practical way, and builds on our ability to treat
not only generalized joint disease by direct gene therapy approaches,
but also focal cartilage injury by gene-enhanced chondrocyte
implantation. Both approaches diminish the likelihood of arthritis,
and may possibly reverse the early stages of arthritis in horses
and other animals.